Summary: Our project focuses on the biology, cell biology, molecular biology and biochemistry of activated lymphocytes, cells that are used for immunotherapy, especially for cancer treatment, and involved in the cellular immune response to xenogeneic tissues. In addition, we have initiated studies examining conditions of expression of porcine endogenous retrovirus under conditions that would be expected to be encountered in clinical xenotransplantation, with particular emphasis on cells of the immune system and factors that regulate them. We have examined the susceptibility of porcine endothelial cells to cytolytic cells of the human immune system. First we investigated how modulation of the redox environment affects the susceptibility of porcine endothelial cells to lysis by IL-2-activated human NK cells. TNF-alpha treatment upregulates the susceptibility of these cells, while treatment of TNF-activated cells with oxidative stress together with nitric oxide reduces the cytotoxicity back to baseline levels. The mechanisms of this reduction appears to involve the transcription factor, nuclear factor kappa B. More recently we have cloned the porcine apoptotic-inducing protein, Fas ligand (CD95L) and found that its transfection into porcine endothelial cells protects them from lysis by human T cells and NK cells. The mechanism appears to be, at least in part, through destruction of the human cytolytic cells by porcine endothelial cells. We have expanded our studies of xenogeneic immune responses in the human anti-pig response in vitro. Our data show that NK cells are the primary mediators of the human vs. pig cytolytic cellular response, and that this response is up-regulated by several human cytokines, including IL-2, IL-12, IL-15, all of which would be expected to be produced in a xenograft recipient. In contrast, IL-18 and IL-8 have no effect. We are continuing to study how NK cells recognize xenogeneic target cells. Finally, we have data to suggest that porcine bone marrow cells spontaneously produce porcine endogenous retrovirus Although this virus may not be infectious for human cells, it appears to be for porcine cells. This finding may be important for the suggested use of bone marrow transplantation from pigs to humans to induce tolerance in potential human recipients of porcine organ grafts.